1. Field of the Invention
The present invention relates to a method of preparing an expression vector which permits simultaneous performance of the expression of retroviral genes and processing after translation, and the resultant plasmid and expression products available by such method of preparation. More particularly, the present invention relates to a method of preparing a plasmid, which causes expression of gag and pol genes of retrovirus by the application of recombinant DNA techniques, causes simultaneous processing of said expression product itself by a protease therein, and mass produces three kinds of core protein encoded by the gag gene, including p17, p24 and p15, and three kinds of enzymes encoded by pol gene, including protease, reverse transcriptase and integrase, individually and independently in the form of mature or active protein molecules, and the plasmid available by said method and expression products thereof. The present invention provides also a plasmid with a high expressing ability of nef gene and Nef protein molecule which is an expression product thereof.
2. Description of Related Art
1. Definition of Retrovirus
Retrovirus is the generic name of viruses classified as belonging to the retrovirus family and characterized by such common features as an envelope, a single-stranded RNA genome, and reverse transcriptase. This virus has a spherical shape having a diameter of 80 to 100 nm, the genome thereof consisting of two molecules of linear (+) stranded RNA with a molecular weight of about 3.times.10.sup.6, these two molecules forming an inverted dimer.
More particularly, the retrovirus family is further classified into the following three subfamilies: oncovirus, lentivirus and spumavirus (R. E. F. Matthews Edt. "Classification and Nomenclature of Viruses-Fourth Report of the International Committee on Taxonomy of Viruses", pp 124-128, S. Karger [Swiss], 1982). Known viruses classed as oncoviruses, also named RNA tumor viruses, include human T cell leukemia virus, feline leukemia virus, murine sarcoma virus, moloney murine leukemia virus, bovine leukemia virus, hog leukemia virus, avian leukemia virus, avian sarcoma virus, avian myeloblastosis virus, and Rous associated virus. Known viruses classed as lentiviruses which are commonly known as viruses causing slow virus infection, include human immunodeficiency viruses types 1 and 2 (hereinafter respectively referred to as "HIV-1" and "HIV-2"), simian immunodeficiency virus, visna virus causing ovine encephalomyelitis, maedi virus causing jaagsiekte, caprine arthritic encephalitis virus, equine infectious anemia virus, and bovine lymphadenitis virus ("Current Topics of AIDS", vol. 1, pp. 95-117, John Wiley & Sons, 1987; Advances in Virus Research, vol. 34, p. 189-215, 1988). The viruses classed as spumaviruses, also named foamy viruses, infect such mammals as humans, monkeys, cattle, and cats. Foamy virus and syncytial virus isolated from these hosts are well known. The term retrovirus as used herein can be taken to include all viruses, known as well as unknown, characterized as retroviruses as described above.
2. Present Situation of Fundamental Research Regarding Retroviral Genes
Retroviruses are important not only from the point of view of the serious and often lethal infectious diseases which they cause in men and other animals, but they are also useful for the understanding of diseases such as sarcoma and for the preparation of material for use in research and genetic engineering. Consequently, much has been published in the literature about these viruses. As is well known, before 1980 retroviruses had been studied, as a model for the oncogenic mechanism, and had been implicated in a new and strange slow virus infectious disease which resulted in incurable secondary diseases. Since the identification of this disease as AIDS in the United States in 1981, comparative studies on various retroviruses have intensively been carried out using the full range of techniques in epidemiology, immunology, virology and molecular biology with a view to establishing methods for the treatment and prevention of AIDS. A huge volume of useful reports concerning AIDS has already been accumulated (Advances in Virus Research, vol. 34, pp. 189-215, 1988; Annual Review of Immunology, vol. 6.pp. 139-159, 1988; Microbial Pathogenesis, vol. 5, pp. 149-157, 1988; "HIV and Other Highly Pathogenic Viruses", pp. 33-41, Academic Press, Inc., 1988; "The control of Human Retrovirus Gene Expression", pp. 79-89, Cold Spring Harbor Laboratory, 1988; Cytological Engineering, vol. 7 (Suppl. 1), pp. S5-S15, 1988).
In the lentivirus the viral genome forms a complex with the reverse transcriptase, the structural protein and a primer tRNA, in the core of the viral particle. The viral genome comprises about ten different genes, including the basic three major genes encoding the viral particle components essential for virus multiplication, i.e., the gag (group-specific antigen) gene encoding the precursor of the core protein, the pol (polymerase) gene encoding a precursor of three different enzymes, and the env (envelope) gene encoding the precursor of the glycoprotein of the envelope. These genes are arranged from the 5' end to the 3' end in the sequence gag, pol, and env. Lentiviruses such as HIV also contain the unusual gene vif, and the genes are arranged in the specific sequence gag, pol, vif (previously called sor), env and nef. Part of the 5' end region of the pol gene overlaps about 240 bases with the 3' end region of the gag gene, and has a different reading frame. Frameshifting is thought to occur during translation of this overlapping portion. After translation of the precursor protein having a molecular weight of 55 kd from the entire aaq region of a total length of about 1.5 kb including that overlapping portion, there occurs cutting, i.e., processing by a protease, and it is considered to become three kinds of protein serving respectively as matrix protein, capsid and nucleocapsid, i.e., in the order of the enumeration given above, p17, p24 and p15. Expression of the entire pol gene region having a total length of about 3 kb produces the above-mentioned enzyme precursor (molecular weight: 100 kd) in the form of a fusion protein capable of being represented as NH.sub.2 --Protease--integrase--COOH, and then, this fusion protein itself is cut or cleaved under the effect of the existing protease produced by the virus or by the protease activity within the same molecule, and this process known as processing is considered to convert it into individual mature proteins (active proteins), i.e., individual enzymes including protease (p12), reverse transcriptase (p66 and p51) and integrase (p32) (Journal of Virology, vol. 62, No. 5, pp. 1808-1809, 1988).
All enzymes mentioned above play important roles in the viral multiplication process and in the process of infection, and the following functions have been confirmed or presumed. The protease participates in post-translational processing, core formation, and the maturation of the viral particle, and is highly specific towards the viruses from which it is derived. Reverse transcriptase is known to have three different enzymatic activities. Thus it functions as an RNA dependent DNA polymerase catalyzing the process of reverse transcription of the genomic RNA into DNA, which is the basic stage of the virus multiplication process. Simultaneously, the ribonuclease H activity of the enzyme specifically digests the RNA strand of the RNA-DNA heteroduplex which is formed, and the DNA dependent DNA polymerase activity produces double-stranded DNA. Reverse transcriptase is popularly used as a tool in genetic recombination. Integrase is an endonuclease acting on the DNA chain, and recognizes and catalyzes the excision of that part of the linear or circular virus double-stranded DNA, which has been transcribed from viral genomic RNA which is to be integrated into the host chromosome DNA, and is thus considered to participate in the process of provirus formation ("HIV and Other Highly Pathogenic Viruses", pp. 33-41, Academic Press, Inc., 1988; "The Control of Human Retrovirus Gene Expression", pp. 79-89; Cold Spring Harbor Laboratory, 1988; Cell Technology (in Japanese), vol. 7 (Suppl. 1), pp. S5-S15, 1988; AIDS Journal (in Japanese), vol. 1, No. 3, pp. 291-300, 1988; Aids, vol. 2 (Suppl. 1), pp. S29-40, 1988; KAGAKU-TO-SEIBUTSU (in Japanese), vol. 27, No. 4, pp. 218-227, 1989).
The region of nef gene having a total length of about 0.4 kb encodes a negative regulatory factor with a molecular weight of about 27 kd, and this regulatory factor is considered to stop multiplication of HIV by acting inhibitively on expression of HIV genome and thus is considered to be associated with latent infection ("Virology", edited by B. N. Fields et al., vol. 2, pp. 1534-1535, published by Raven Press [U.S.], 1990).
3. Present Status and Problems in Mass production of Retroviral Gene Products by Genetic Engineering
Mass production of gene products (hereinafter referred to as "antigens") is widely conducted at various places in the world for the purpose of developing vaccines for human beings and animals and for the purpose of developing diagnostic drugs. As a typical example, the process for mass production of antigens of AIDS virus is outlined below. Principal antigens under study for mass production include precursor glycoprotein gp160 of env gene and processed products thereof, gp120 and gp41 ("Forefront of Countermeasures Against AIDS", edited and authored by Toshiaki Komatsu, vol. 2, pp. 477-495, published by CMC, 1989). For the production of gp160, for example, use is reported of vacuolovirus vector and insect cells (Proceedings of National Academy of Sciences (USA), vol. 84, pp. 6924-6928, 1987), recombinant vaccinia virus and BSC-40 or Hela cells (Nature, vol. 320, pp. 537-540, 1986; Nature, vol. 330, pp. 259-262, 1987), and adenovirus vector and A549 cells ("Vaccine 89", pp. 207-217, Cold Spring Harbor Laboratory, 1989). There have also been reported the expression of gp120 gene region by means of plasmid inserted and linked with that region and CHO cell line (Science, vol. 233, pp. 209-212, 1986), and production of gp120 with Escherichia coli (Science, vol. 234, pp. 1392-1395, 1986). In addition, known techniques regarding mass antigen production are classified and briefly listed below (hereinafter European Patent Provisional Publication No. is abbreviated as [EP], West German Patent Provisional Publication No. as [DE], United States Patent Application No. as [US], PCT International Patent Publication No. as [Wo], and French Patent Provisional Publication No. as [FR], respectively):
(1) Techniques aimed at mass expression of such genes as gag, pol, and env (hereinafter described for each host employed): PA1 (2) Techniques for producing recombinant vaccinia virus inserted with such genes as gag, pol and env: PA1 (3) Techniques for causing expression of env gene by virus vector: PA1 (4) Techniques for causing expression of AIDS virus antigen as a fusion protein with hepatitis B virus antigen:
Escherichia coli (EP 331961, EP 322394, DE 3727137, DE 3724016, EP 293792, US 07/218304, US 07/110348, EP 255190, WO 87/07296, DE 3711016, EP 227169, EP 199301, EP 219106): PA2 Yeast (DE 3804891, EP 322394,); PA2 Japanese Patent Provisional Publication No. 1-148183, FR 2620030, FR 2607518, FR 2600079, FR 2587720, FR 2596711, EP 256677, WO 87/06260, WO 87/06262; PA2 Use of polyoma virus: Japanese Patent Provisional Publication No. 1-39991 and PA2 vacuolorivus: EP 272858, EP 265785; and PA2 EP 278940
Apart from the production technique of recombinant virus applicable as an effective component of a live vaccine, for example, three elements including mass expression, secretion of gene products outside the host, and processing after translation are very important with a view to ensuring a low production cost and a high quality in industrial production of useful proteins by means of an expression vector. It is necessary to consider these three elements in constructing an expression vector. As is clear from the outline of the prior art techniques described above, mass expression has been studied by various researchers, and development of a secretory production system (Nippon Nogeikagaku Kaishi (in Japanese), vol. 60, No. 5, pp. 1035-1063, 1990) is widely and actively in progress. However, little attention is given by contrast to the contrivance of processing after translation despite its importance in saving labor in the refining process of gene products and improving purity of such products. It is therefore evident that development of an expression system allowing processing after translation has a valuable significance. Since Nef protein is useful in the field of diagnosis of AIDS and clarification of crisis mechanism, and therapy from the point of view of its antigenicity and functions, establishment of a mass production technique is expected.